Part:BBa_K2717013:Experience

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Applications of BBa_K2717013

It has been experimentally verified that the part can successfully express the VHb protein and transport the protein to the periplasmic space of E. coli. After the growth curve measurement experiment, the bacteria expressing the part had higher environmental capacity than the same strain expressing the empty plasmid, and the plateau was longer. We also designed a vector containing only VHb. Experiments have shown that the strain expressing TorA and VHb has a higher environmental capacity and a faster growth rate than the strain expressing VHb.

We gain VHb directly form official Kit(BBa_K1321200) and gain TorA signal peptide by company synthesis. Then VHb and TorA signal peptides overlap to a complete coding region and be infused to our modified pUC19, just after lac promoter and RBS B0034. New plasmid is transformed into strain E. coli BL21-Stbl3. We also design another Stbl3 with plasmid contains VHb but without TorA signal peptide as control. We select single colony from each plate culture medium then inoculate them into 20 ml LB fluid medium containing 0.1% antibiotics (except for wild type). Culture them in a shaking table at 37℃ overnight, then use M9 fluid medium to dilute them as OD600 reach 0.01. Here, each strain is separated into two kinds of growth mode. Aerobic growth was conducted using 50 ml flasks containing 10 ml of culture medium under rotary shaking at 200 rpm; microaerophilic growth was conducted using a 50 ml flask containing 100 mL of culture medium under rotary shaking at 100 rpm. Collect 200μl bacteria solution to 96well plate each 4 hours use microplate reader to monitor its OD600. Under aerobic condition, bacteria grow faster after adding TorA signal peptide, and the K value has improved. As for microaerophilic environment, bacteria can maintain stable growth trend for a long time after adding TorA signal peptide. All these results suggest that our new part can make VHb work better than before and give bacteria growth advance.

Fig.2 Growth Curve for VHB and Tor-VHB

Figure.3 SDS-Page gel result of periplasm protein.

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